The goal of the proposed research is to characterize the protease that converts the carboxypeptidase H (CPH) precursor, pro-CPH, to the smaller mature CPH, and to understand how this protease is regulated in the tissue-specific processing of pro-CPH. These studies are important because CPH is necessary for the regulation of enkephalin opiate peptide production via cleavage of the inactive proenkephalin. Recent studies by the PI show that pro-CPH itself must undergo post-translational cleavage and modification to form catalytically active enzyme. Furthermore, the extent of pro-CPH processing differs in adrenal medula compared to posterior pituitary (and other tissues), indicating that the protease that cleaves pro-CPH is regulated in a tissue-specific manner. However, the pro-CPH cleaving protease has not been extensively studied. Therefore, this proposal will characterize the pro-CPH cleaving activity in secretory vesicles of bovine adrenal medulla and pituitary, using authentic 35S-(Met)-pro-CPH as substrate for in vitro activity assays. Kinetics of product formation by this proteolytic activity will be determined by cellular 35S-methionine pulse-chase studies in adrenomedullary chromaffin cells. For regulatory studies of this protease at the post-translational and transcriptional levels, specific antibodies as probes for enzyme protein and cDNA probes for examination of enzyme gene expression will be produced. This will first require enzyme purification and its biochemical characterization, including partial amino acid sequence determination for design of oligonucleotide probes for cDNA molecular cloning. Purified enzyme will be used as antigen for production of specific antibodies (polyclonal and monoclonal). Regulation of the pro-CPH cleaving protease in adrenal medulla compared to posterior pituitary will be studied with respect to characteristics of enzyme activity, molecular forms of enzyme protein, and gene expression. The studies will define the regulatory role of the pro-CPH cleaving protease in the tissue-specific processing of pro-CPH. The new findings from these studies can be extrapolated to neuroendocrine systems to demonstrate the intimate role of a prohormone processing enzyme such as CPH in the formation of enkephalins and other peptide neurotransmitters and hormones. An understanding of the mechanisms controlling the production of these potent, physiologically active peptides should be valuable in elucidating molecular defects underlying some neurological disorders, providing new insight into therapeutic strategies.